Polymorphism of the Rdna Chromosomal Regions in the Weatherfish Misgurnus Fossilis (Teleostei: Cobitidae)

ISSN 0015-5497, e-ISSN 1734-9168 Folia Biologica (Kraków), vol. 65 (2017), No 1 https://doi.org/10.3409/fb65_1.63

Polymorphism of the rDNA Chromosomal Regions in the Weatherfish Misgurnus fossilis (Teleostei: Cobitidae)

Aneta SP[Z, Alicja BOROÑ, Konrad OCALEWICZ, and Lech KIRTIKLIS

Accepted February 01, 2017 Published online April 24, 2017 Published April 28, 2017

SP[Z A., BOROÑ A., OCALEWICZ K., KIRTIKLIS L. 2017. Polymorphism of the rDNA Chromosomal Regions in the Weatherfish Misgurnus fossilis (Teleostei: Cobitidae). Folia Biologica (Kraków) 65: 63-70.

The weatherfish, Misgurnus fossilis (Linnaeus, 1758), is native and an endangered species in Europe. Individuals with a chromosome number of 100 representing the most frequent cytotype seem to have a polyploid origin in comparison with rarely observed individuals of 50 chromosomes. This study cytogenetically characterized M. fossilis (five males, seven females) possessing 100 chromosomes to show the chromosomal distribution of major and minor ribosomal DNAs, and telomeric DNA repeats using fluorescence in situ hybridization with 28S and 5S rDNAs, and telomere peptide nucleic acid probes, respectively. Silver nitrate (AgNO3) staining was used to show the activity of nucleolus organizer regions (Ag-NORs) and chromomycin A3 (CMA3) fluorochrome staining to detect the GC-rich chromosome regions. In all individuals size polymorphism of Ag-NORs and rDNA clusters was identified. Most of the studied individuals exhibited four 28S rDNA sites found in the short arms of the submetacentric and subtelo-acrocentric chromosomes, however in one female only two 28S rDNA sites were observed. Chromosome regions consisting of 28S rDNA corresponded to AgNO3 positive and GC-rich chromatin. The 5S rDNA loci were located predominantly in a pericentromeric position of eight or six subtelo-acrocentric chromosomes and did not co-localize with 28S rDNA. The telomeric repeats were exclusively localized at the ends of all M. fossilis chromosomes. The presence of four NOR-bearing chromosomes may reflect the polyploid origin of M. fossilis with 100 chromosomes. In turn, reduction of the NOR sites observed in one specimen might be considered as part of the rediploidization process.

Key words: Cytogenetics, NORs, FISH, polyploidy, ribosomal genes, telomeres.

Aneta SPÓZ, Alicja BOROÑ, Lech KIRTIKLIS, Department of Zoology, Faculty of Biology and Biotechnology, University of Warmia and Mazury in Olsztyn, M. Oczapowskiego 5, 10-718 Olsztyn, Poland E-mail: [email protected] Konrad OCALEWICZ, Department of Marine Biology and Ecology, Faculty of Oceanography and Geography, University of Gdansk, Marsza³ka Pi³sudskiego 46, 81-378 Gdynia, Poland.

The weatherfish, M. fossilis (Linnaeus, 1758), is Individuals of M. fossilis distributed throughout the only Misgurnus species native to Europe but Europe rarely show a diploid number of 50 chro- presently exhibiting a Eurasian distribution (KOT- mosomes (VUJOSEVIC et al. 1983) as the karyo- TELAT &FREYHOF 2007). It is a freshwater (or type of 100 chromosomes is the most frequently rarely brackish water) bottom-dwelling species observed cytotype (RAICU &TAISESCU 1972; considered endangered in Europe due to the loss of BOROÑ 2000; ENE &SUCIU 2000). In two locali- habitat (FREYHOF 2011). ties in the Czech Republic individuals of different Available karyological data for Misgurnus spe- ploidy level were reported (PALÍKOVÁ et al. 2007; cies revealed diploid chromosome numbers from DROZD et al. 2010) (Table 1). 2n=48(LEE et al. 1987; KIM & PAK 1995) and 2n = 50 M. anguillicaudatus individuals with 50 and 100 chro- (VASIL’EV &VASIL’EVA 2008) to species of poly- mosomes are recognized as diploids and tetraploids, ploid origin, e.g. M. anguillicaudatus and M. fossilis respectively (e.g. LI et al. 2010, 2013; ARAI & (BOROÑ 2000; ENE &SUCIU 2000; LI et al. 2010; FUJIMOTO 2013). Similarly, it has been proposed ARAI &FUJIMOTO 2013). that the M. fossilis lineage composed of individu-

Ó Institute of Systematics and Evolution of Animals, PAS, Kraków, 2017 Open Access article distributed under the terms of the Creative Commons Attribution License (CC-BY) OPEN ACCESS http://creativecommons.org/licences/by/4.0 64 A. SP[Z et al. als with 100 chromosomes appeared in the course 195 mm, respectively) were collected from two of the ancestral polyploidization event (DROZD et al. sites in Poland. Seven of them (three males and 2010). Such a karyotype may still be arranged into four females) were collected from the Kortowka 25 quadruplets (ENE &SUCIU 2000; PALÍKOVÁ et al. River tributary of Kortowskie Lake (53°45¢43"N; 2007) however such individuals behave as normal 20°26¢42"E), the Pregola River drainage (Baltic Sea diploids showing regular gametogenesis(BOROÑ basin). The other five specimens (two males and 2000). Moreover, induced gynogenetic progeny three females) were caught from the Siemianowka with only a maternal set of chromosomes (50) was Dam Reservoir (52°55¢34"N; 23°49¢39"E) at the inviable (ARAI &FUJIMOTO 2013), indicating a Narew River, Vistula River drainage (Baltic Sea progressive process of genome diploidization fol- basin). Taxonomic identification of individuals lowing the polyploidization event (MABLE et al. was established based on their external morpho- 2011; WERTHEIM et al. 2013). In contrast to M. fossilis, logical features according to KOTTELAT and induced gynogenetic progeny of M. anguillicauda- FREYHOF (2007). tus with 50 chromosomes were viable indicating Fish sampling and valid animal use protocols that individuals with 100 chromosomes are geneti- for experiments were done with the permission cally true tetraploids (LI et al. 2012). Therefore of the Polish Ministry of Environment (no. DOP- M. fossilis with 100 chromosomes should be treated OZGiZ.6401.10.12.2011.ls) and the Local Ethics as functionally diploid (ARAI &FUJIMOTO 2013). Commission from the University of Warmia and Diploidization as the gradual conversion of poly- Mazury in Olsztyn, Poland (no. 20/2013/N). ploidy to diploidy takes place through chromo- Voucher specimens were preserved frozen at the some rearrangements and retention or loss of the Department of Zoology, University of Warmia duplicated genes (LIEN et al. 2016). Such genome and Mazury in Olsztyn, Poland. reorganization may also affect activity and distribution of the ribosomal genes and telomeric DNA Chromosome preparation and conventional sequences commonly used as very informative cy- staining togenetic features (FUJIWARA et al. 1998; ARAI & FUJIMOTO 2013; GORNUNG 2013; OCALEWICZ et Mitotic chromosome preparations were made al. 2013; BISCOTTI et al. 2015). In higher eukaryo- from each individual following BOROÑ (2000). tes, ribosomal RNA genes (rDNAs) are organized First, fish were injected with a dose of 1 ml of into the nucleolus forming major rDNA (45S) 0.05% colchicine solution per 100 g body weight family composed of clusters of multiple copies of to prevent microtubule formation during cell divi- tandemly repeated units with coding regions for sion to arrest the chromosomes in metaphase. 18S, 5.8S and 28S rRNA genes and the non- Then, fish were anaesthetized using MS 222 prior nucleolus forming minor rDNA (5S) family to sacrifice. Mitotic chromosomes were obtained (FUJIWARA et al. 1998). from kidney cell suspensions using the air-drying Cytogenetic data for M. fossilis is restricted to method. The kidney cells were exposed to a hypo- conventional staining techniques(BOROÑ 2000; tonic solution (0.075 M KCl) for 30 min and fixed ENE &SUCIU 2000). Thus, in the present paper, in methanol: acetic acid (3:1). After drying at room we performed a molecular cytogenetic study on temperature, chromosomes were stained with a so- M. fossilis with 100 chromosomes to analyze the lution of 4% Giemsa (pH=6.8). chromosomal distribution of ribosomal genes and Chromosome slides of all studied individuals telomeric DNA sequences by fluorescence in situ were sequentially stained with silver nitrate hybridization (FISH) with 28S rDNA, 5S rDNA (AgNO3) and chromomycin A3 (CMA3) fluoro- and telomeric DNA repeats and to show the chro- chrome, according to SOLA et al. (1992) to detect mosomal distribution of active nucleolar organizer respectively, the active NOR (Ag-NORs) and the regions (Ag-NORs) and GC-rich sites using se- GC-rich chromosome regions. quential staining with silver nitrate (AgNO3)and chromomycin A3 (CMA3) fluorochrome. The results provided information about the functional struc- Probes and fluorescence in situ hybridization ture of the chromosomes and are discussed in the (FISH) context of tetraploidization and re-diploidization Double-color FISH with human 28S and loach processes in the genome of this polyploid species. 5S rDNA probes was used according to FUJIWARA et al. (1998) and BOROÑ et al. (2009). The 28S rDNA probe was labeled with digoxigenin-11-dUTP us- Material and Methods ing the DIG-Nick Translation Mix kit (Roche), while the 5S rDNA probe was labeled with biotin- In total 12 individuals, five males and seven fe- 16-dUTP using Biotin-Nick Translation Mix kit males of M. fossilis (average length of 170 mm and (Roche), according to the manufacturer’s instruc- rDNAs Chromosomal Polymorphism in Weatherfish 65 tions. The chromosome slides were pre-treated metaphase plates, being the largest elements in the with RNase for 60 min at 37°C in a moist chamber. chromosome complement (Fig. 1a-b). One of the After denaturation for 1 min in 70% formamide homologues of the submetacentric chromosome (FA)/2×SSC, chromosome slides were dehydrated pair no. 15 clearly evidenced a secondary structure in a cold (-20°C) ethanol series, 70% for 5 min and along its short arm easily distinguishable after 80%, 90%, and 100% for 2 min each. Hybridiza- Giemsa staining while this structure was less evi- tion was performed at 37°C in a moist chamber dent in the other homologue (Fig. 1a-b, shown in- with a mixture containing denatured rDNA probes, boxed). Bovine Serum Albumin (20 mg/ml), 50% dextran After Ag-NO3 staining, four Ag-NORs were iden- sulphate, 20×SSC, and double-deionized water. tified in the short (p) arms of two sm (pair no. 15) Post-hybridization washes were performed in 50% and two sta chromosomes (pair No. 35) in a sub- FA/2×SSC at 37 °C for 20 min, 2×SSC and 1×SSC for centromeric position in 87 (83.7%) out of 104 ana- 20 min each, and 4×SSC for 5 min. 28S and 5S rDNA lyzed metaphase plates in 11 out of 12 individuals probes were detected with Anti- Digoxigenin- of M. fossilis (Fig. 1c).Two (11.5%) and three Rhodamine (Roche) and Avidin-Fluorescein (Roche), (4.8%) NORs were observed in the remaining respectively, and chromosomes were counterstained metaphases. Sequential AgNO3 and CMA3 stain- with DAPI in Antifade solution (Vector Laborato- ing confirmed the co-location of NORs and ries, USA). CMA3-positive sites (Fig. 1c-d). Among analyzed Telomeric DNA repeats were detected by FISH metaphases, NORs exhibited length polymor- using a telomere Peptide Nucleic Acid (PNA) phism related to variation in NOR size (both Ag- probe conjugated with FITC (DAKO, Denmark) and CMA3-positive sites) (Fig. 1c). according to the manufacturer’s protocol. Both A different pattern with only two NORs located chromosomal DNA and PNA probes were dena- on the short arms of two sm chromosomes (pair tured at 85°C for 5 min simultaneously. Hybridiza- No. 15) was found in most of the metaphases tion took place in darkness at room temperature for (92.3%) of one female from Kortowka River 60 min. Metaphase spreads were counterstained (Fig. 2a). This was detected by both Ag- and with DAPI in antifade solution Vectashield (Vector, CMA3-staining (Fig. 2a-b). However, the single Burlingame, USA). pair of NORs of this female exhibited the same length polymorphism related to variation in NOR Microscopy and Image Processing size (Fig. 2a-b). In the remaining 7.7% of the metaphases only one positive site was observed. At least 15-20 metaphase spreads from each individual were analysed using a Nikon Eclipse 90i fluorescence microscope equipped with ProgRes Major rDNA mapping MFcool camera (Jenoptic) and supported by an ap- FISH with the 28S rDNA probe analyzed in propriate filter set. The images were processed us- 166 metaphase plates of six (out of seven) females ing Lucia software 2.0 (Laboratory Imaging). and five males confirmed data from Ag-NO3 and All chromosomes were classified according to CMA3 staining and revealed four loci in their chro- LEVAN et al. (1964), where metacentric (m) and mosome complement in the majority of the meta- submetacentric (sm) chromosomes were consid- phase plates (83.7%) (Fig. 1e-g). Positive signals ered as biarmed, whereas subtelocentric and acro- were found in the short arms of two sm (pair No. 15) centric (sta) as uniarmed elements. and two sta chromosomes (pair No. 35) (Fig. 1f-g). Positive hybridization on sm chromosomes were commonly intense and large, whereas the signals Results on the sta chromosome pair were smaller and weaker (Fig. 1e-g). In the remaining 16.3% of metaphase plates, the 28S rDNA loci were located Karyotype and banding patterns in three (11.4%) or two (4.9%) chromosomes (data Figure 1a-d and Figure 2a-b present karyotype not shown). No variability of 28S rDNA location and banding patterns of M. fossilis. Analyzed indi- was found between males and females from either viduals showed a diploid chromosome number of of the two localities. The size polymorphism ob- 100 in 168 (93.3%) out of 180 analyzed metaphase served in chromosome pair no. 15 after conventional plates. In the remaining 6.7% of metaphases, 98 staining was less visible after FISH (Fig. 1f-g). and 99 chromosomes were observed. The karyo- Two distinct heteromorphic 28S rDNA hybridi- type was composed of 16m + 20sm + 64st/a chro- zation signals on the short arms of two sm chromo- mosomes (Fig. 1a-b) and thus a chromosome arm somes (pair No. 15) were observed in 92.3% of the number (NF) of 136. The four submetacentric 26 metaphases examined in the single female from pairs nos. 9-12 were easily recognizable in all Kortowka River with only two NORs (Fig. 2c-e). 66 A. SP[Z et al.

Fig. 1 Giemsa-stained metaphase spread (a) and respective karyotype (b) with in-boxed pair evidencing NOR secondary structure. The most common cytotype of Misgurnus fossilis from Poland evidenced by silver-stained metaphase (c) and CMA3-stained metaphase (d), FISH with 28S (red signals, red arrows) and 5S (green signals and arrows) rDNA probes shown in a metaphase plate (e), assembled karyotype (f) and ideogram (g); two pairs of chromosomes with Ag-NORs (top row) sequentially chromomycin A3-stained (middle row) and their ideogram are shown in the inset in c. Bar = 10 µm. rDNAs Chromosomal Polymorphism in Weatherfish 67

Fig. 2 Metaphase spread of the single female individual of Misgurnus fossilis from Kortowka River with a different cytotype evidenced by silver-stained metaphase (a), CMA3-stained metaphase (b) and FISH with 28S (red signals, red arrows) and 5S (green signals and arrows) rDNA probes shown in a metaphase plate (c), assembled karyotype (d) and ideogram (e); one pair of chromosomes with Ag-NORs (top row) sequentially chromomycin A3-stained (middle row) and its ideogram (bottom row) is shown in the inset in a. Bar = 10 µm.

Fig. 3. Metaphase spread of M. fossilis showing the chromosomes after DAPI staining (a) and PNA-FISH with telomeric probe (b). Bar = 10 µm. 68 A. SP[Z et al.

In the remaining metaphases (7.7%) a single 28S nome duplication event in this lineage of M. fossilis. rDNA cluster was observed. In this female how- Similar studies performed on individuals with ever, the length polymorphism of the NOR was 50 chromosomes would be useful for comparison, also conspicuous after FISH (Fig. 2d-e). but they seem to be rare (Table 1) and we did not have such samples. Minor rDNA mapping While 28S rDNA sequences in M. fossilis of 100 chromosomes were exclusively mapped to the FISH with 5S rDNA probe revealed eight (52.1%) short arms of two sm and two st chromosomes, (Fig. 1e-g) and six (35.8%) positive signals in all those in M. anguillicaudatus were detected in two individuals analyzed. Minor rDNA clusters were and four homologous chromosomes in diploids located at the pericentromeric positions of sta chro- (2n =50) and tetraploids (4n = 100), respectively mosomes of pairs Nos. 22, 26, 33 and 48 (Fig. 1f-g). (LI et al. 2010). This distribution of NORs sug- The remaining metaphases (23 out of 190) re- gests the duplication of the entire M. anguillicau- vealed four (3.7%) or seven (8.4%) 5S rDNA loci datus genome from diploid to tetraploid status (LI with intra-individual variation. Hybridization sig- et al. 2010, 2011). Two morphologically different nals had different intensities on various chromo- pairs of NOR-bearing chromosomes in M. fossilis somes and could be classified as strong or weak. may be connected with its allotetraploid origin, but Usually, in the metaphase plates with eight 5S rDNA the formation of tetraploidy still requires many signals, four signals were stronger (pairs nos. 22 studies (LI et al. 2010). and 26 in Fig. 1f-g) than the others (pairs nos. 33 and 48 in Fig. 1f-g). These weak signals were not Although two pairs of NOR bearing chromo- visible in the metaphase plates with six hybridiza- somes seem to be the typical pattern for M. fossilis, tion sites of 5S rDNA (data not shown). the number of active NORs (Ag-NORs) varied from two to four (BOROÑ 2000; present work) and The M. fossilis female characterized by only two from three to four (ENE &SUCIU 2000) consistently NORs revealed the same distribution pattern and with different expression activity of the rRNA number of 5S rDNA loci as all other individuals genes in the preceding interphase (REEDER 1990). analyzed (Fig. 2c-e). Only two clusters of 28S rDNA in one female Both classes of rDNA probes were always lo- suggests that the other two clusters putatively cated on different chromosomes (Fig. 1e-g and present at the subtelocentric chromosomes were Fig. 2c-e). No variability in the 28S and 5S rDNA composed of copy numbers too low to be detected. hybridization signals between individuals from The different intensity of FISH signals between sm the two localities as well as no sex-dependent vari- and sta and NOR size polymorphism observed in ability in the cytogenetic features was found. the karyotype of M. fossilis agree with the results exhibited in the tetraploid M. anquillicaudatus and Mapping of telomeric sequences are presumably also related to different copy numbers of the rDNAs as a result of unequal crossing The telomeric repeats were exclusively local- over (LI et al. 2010). ized at the ends of all M. fossilis chromosomes in all the individuals. No interstitial signals were ob- On the other hand we cannot exclude that these sites served (Fig. 3) and no differences in the distribu- of major rDNAs/NORs were eliminated by the re- tion of the telomere signals between specimens or duplication process to re-establish the diploid-like between males and females from the two localities condition. Examination of fishes of tetraploid re- were found. vealed that both duplicated gene copies may be maintained and retain original functions (1), one of the copies may attain a new function (2) or the duplicated gene may be suppressed and lost (3) (LI et Discussion al. 2015; LIEN et al. 2016). If so, the single NOR The physical mapping of 28S rDNA revealed for locus found in one of the examined M. fossilis the first time that the karyotype of M. fossilis from could suggest a loss of the duplicated NOR. the lineage with 100 chromosomes is characterized Minor rRNA genes of M. fossilis showed great by four independent major rDNA clusters. Their variability in terms of cluster size and genomic dis- distribution corresponded to Ag- and CMA3-pos- tribution. Some other Cypriniformes species also itive sites (BOROÑ 2000; ENE &SUCIU 2000; pres- demonstrate multiple locations of 5S rDNA clusters, ent study). Taking into account that a single pair of e.g. C. taenia (BOROÑ et al. 2006) and Carassius NORs is considered as the plesiomorphic condi- carassius (SPOZ et al. 2014). A single pair of 5S rDNAs tion in fishes (MARTINS &WASKO 2004; NAKA- is considered as the ancestral condition in fishes JIMA et al. 2012; GORNUNG 2013), two pairs of (MARTINS &WASKO 2004), so multiple 5S rRNA NOR chromosomes may confirm the ancestral ge- gene clusters observed in M. fossilis seems to be rDNAs Chromosomal Polymorphism in Weatherfish 69 a derived state. The DNA sequences encoding 5S rRNAs BOROÑ A., OZOUF-COSTAZ C., COUTANCEAU J-P., WORO- were located in the distal position in the chromo- NIECKA K. 2006. 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